The purpose of this project is to determine the role that repetitive elements (REs) play in the biological outcome of environmental exposures. While it is known that the expression of REs changes in response to environmental agents, mechanistic insights into the impact of REs on the biology of cells and organisms is an area of research that has not been explored in depth. We are specifically interested in studying the extent to which REs alter the expression of adjacent genes through the formation of fusion transcripts (FTs). We chose to use RNA-seq to study this problem, and we have developed a robust analytical pipeline to detect FTs. In our initial study, we have found that many genes in the brain of mice are expressed as FTs, including the peroxisome proliferator gamma co-activator 1 (Pgc1). Pgc1 is a master metabolic regulator that was identified back in 2004 as a co-transcriptional activator of the mitochondrial biogenesis program. Our analytical strategy identified two repeat-containing isoforms of Pgc1 in the mouse brain: one involving a simple sequence repeat (SSR), about 500 Kb upstream from the canonical promoter that spliced to the second coding exon of the gene. The second fusion isoform involved the same SSR that spliced to a SINE (small interspersed nuclear element), that is about 250 Kb downstream from it, and then spliced to the second coding exon. Analysis of limited publicly available RNA-seq data sets revealed that both of these new FT isoforms are brain specific. Moreover, within the brain, the SSR-SINE-exon 2 isoform seems to be confined to neurons, while we detected the SSR-exon 2 isoform only in oligodendrocytes. We also analyzed publicly available ribosomal profiling data sets and found evidence that the SSR-containing isoforms are actively translated in the brain. Additional support that these new FTs make proteins come from our work in which the cloned SSR-SINE-exon 2 isoform containing a myc tag were found to give rise to proteins of the expected size on a western blot. In this past year, we have contracted with a company to generate antibodies to the amino-termini of the proteins that we predict are specific for the predicted FT isoforms of the gene. Our initial analysis demonstrated that the antibodies that recognize the C-terminus, or the second coding exon, are very specific. We are still working on optimization of the work with the antibodies, but we have been able to demonstrate that an antibody to the protein predicted to be expressed from the SSR-SINE-exon2 FT does not produce a protein in homozygous mutant animals that we generated using the CRISPR-cas system (see below). We have generated mice that specifically mutate the two FT expressing Pgc1 in the brain by creating deletions in either the SSR or the SINE using the CRISPR/cas9 technology. While the former should ablate the two brain isoforms, the SINE-mutation should abolish only the isoform expressed in neurons. Because neither of these isoforms is expressed in other tissues, we expect to be able to dissect the role of these FTs in the brain. Most of the effort over the past year has involved studying one of the SINE mutations, which carries a 4 bp deletion that is just downstream from the predicted ATG in the SINE element. Detailed analysis of the brains of the mutant mice revealed no obvious pathological defects and the animals do not show any specific visible phenotypes over time (animals are now about 1 year old) under normal housing conditions. We have performed a series of behavioral tests in the animals, including the water maize, open field and rotarod. While no changes were observed in most tests, the mutant animals show dramatic phenotype associated with the rotarod test. This prompted us to evaluate gene expression profiles in the cerebellum of the mutant mice, since this region of the brain has been linked to motor and coordination defects that are detectable with the rotarod test. We determined that the SSR-SINE-exon2 isoform is the principal form of Pgc1 expressed in the cerebellum. Unexpectedly, we found substantial up-regulation of a number of genes in the cerebellum of the mutant animals, many of which are involved in neuronal synapse or neurotransmitter metabolism or transport. Just a few genes were downregulated, suggesting that this new brain-specific isoform of Pgc1a may function as a co-transcriptional repressor (instead of activator) for specific genes in the cerebellum and possibly in other parts of the brain. We have validated the differential expression of a number of these genes by real time-quantitative PCR and are now in the process of validating these findings in a set of independent animals.